Presence of hepatitis E virus in testis of naturally infected wild boars

Abstract The hepatitis E virus (HEV) is the main cause of viral acute hepatitis in the world, affecting more than 20 million people annually. During the acute phase of infection, HEV can be detected in various body fluids, which has a significant impact in terms of transmission, diagnosis or extrahepatic manifestations. Several studies have isolated HEV in the genitourinary tract of humans and animals, which could have important clinical and epidemiological implications. So, our main objective was to evaluate the presence of HEV in testis of naturally infected wild boars (Sus scrofa). For it, blood, liver, hepatic lymph node and testicle samples were collected from 191 male wild boars. The presence of HEV was evaluated in serum by PCR, as well as in tissues by PCR and immunohistochemistry. Four animals (2.09%; 95%CI: 0.82–5.26) showed detectable HEV RNA in serum, being confirmed the presence of HEV‐3f genotype in three of them by phylogenetic analysis. HEV was also detected in liver and/or hepatic lymph nodes of the four animals by RT‐PCR, as well as by immunohistochemistry analysis. Only one of these wild boars also showed detectable viral load in testis, observing HEV‐specific labelling in a small number of fibroblasts and some Sertoli cells. Our results confirm the presence of HEV genotype 3 in naturally infected wild boar testis, although no associated tissue damage was evidenced. This study does not allow us to discard semen as a possible source of HEV transmission in suids. Future experimental studies are necessary to evaluate the impact of HEV genotype 3 on fertility and the possibility of transmission through sexual contact in this specie.

Culture and Sport, Grant/Award Numbers: FPU17/01319, FPU19/03969; European Social Fund, Grant/Award Number: 2018/12504 presence of HEV genotype 3 in naturally infected wild boar testis, although no associated tissue damage was evidenced. This study does not allow us to discard semen as a possible source of HEV transmission in suids. Future experimental studies are necessary to evaluate the impact of HEV genotype 3 on fertility and the possibility of transmission through sexual contact in this specie.

K E Y W O R D S
genotype 3, hepatitis E virus, testicle, wild boar

INTRODUCTION
Hepatitis E virus (HEV) belongs to the genus Orthohepevirus (family Hepeviridae), including Paslahepevirus balayani species the most important strains for human and animal health (Purdy et al., 2022). HEV is the main cause of viral acute hepatitis in the world, affecting more than 20 million people annually (WHO, 2017).
Eight genotypes (1 to 8) of HEV have been described, of which only genotypes 1 and 2 exclusively affect humans and are associated with typical epidemics of emerging countries, while the genotypes 3 and 4 have been isolated in a wide range of animals and are the main cause of sporadic autochthonous cases of hepatitis E in industrialized countries (Denner, 2019;Meng, 2013;Nimgaonkar et al., 2018).
Here, hepatitis caused by HEV is endemic with the appearance of small sporadic outbreaks (Purcell & Emerson, 2008), where the liver is the main affected organ, although other extrahepatic manifestations have also been observed (Horvatits & Pischke, 2018). Transmission of genotypes 3 and 4 is mainly zoonotic, through direct contact or consumption of raw or undercooked pork or game meat products or shellfish (Faber et al., 2018;Syed et al., 2018;Wang et al., 2019); nevertheless, other forms of contagion have also been demonstrated, such as blood transfusions and organ transplantation (Domanović et al., 2017;McPherson et al., 2018). Although these sources of transmission have been well documented in different studies and are considered the main widespread routes, other possible transmission routes have also been suggested.
The efficiency of sexually transmitted HEV infection and its impact on fertility is unknown. There are only few reports on the presence of HEV RNA in human semen (Huang et al., 2018;Li et al., 2019;Soomro et al., 2017) and in reproductive organs of mice and suids experimentally infected (Schlosser et al., 2014;Situ et al., 2020). However, in the animal experimental cases, the infection was established intravenously, where distribution of the virus is always greater. Furthermore, histological analysis revealed damage on testicular tissue in humans and mice, suggesting that viral replication is possible at this anatomical site (Huang et al., 2018;Situ et al., 2020) At post-mortem examination carried out at the meat board, a whole blood sample was taken from all the animals using puncture of the cavernous sinus of the dura mater (Arenas- Montes et al., 2013). Blood samples were centrifuged at 700 g for 10 min to obtain the serum, which was frozen at -80 • C until analysis. Samples of liver, hepatic lymph nodes and testis were also collected from all the animals. The tissue samples were divided in two, a piece of each was submerged in RNAlater ® Stabilization Solution (Thermo Fisher Scientific Inc., Waltham, MA, USA) and frozen at -80 • C until analysis, and the other piece was fixed by immersion in 10% buffered formalin solution for histopathological and immunohistochemical study.

Histopathological and immunohistochemical examination of tissues for HEV
The formalin-fixed samples were dehydrated with a graded series of alcohols, cleared in xylene and embedded in paraffin wax. After making 4 µm sections and staining with haematoxylin and eosin (H&E), two blinded and experienced observers then performed a histopathological evaluation of the tissue sections.
Sections of the formalin-fixed paraffin-embedded tissue samples (3 µm) were routinely processed for immunohistochemistry (IHC) using the avidin-biotin-peroxidase complex (ABC) method described by Risalde et al. (2017). Briefly, endogenous peroxidase activity was exhausted by incubation of the samples with 0.3% hydrogen peroxide in methanol for 30 min at room temperature (RT, approx. Laboratories, Burlingame, CA) and counterstained with haematoxylin.
As positive controls, liver and hepatic lymph node samples from a pig experimentally infected with HEV genotype 3 were used, confirmed by real-time RT-PCR and IHC (samples from the study of Schlosser et al., 2014). As negative controls, wild boar tissues without evidence of HEV-RNA in serum or randomly selected tissues were used. More-over, rabbit non-immune sera (Vector Laboratories, Burlingame, CA) were used in place of specific primary antibody as additional negative controls.
The evaluation of the immunohistochemical examination in the organs selected was performed for the presence of target cells to anti-HEV antibody, classifying the organs as without staining (-), and with scarce (+), moderate (++) or high (+++) number of immunolabelled cells.

Statistical analysis
The prevalence of HEV was determined by the coefficient of positive animals/total animals tested, using two-sided exact binomial 95% confidence intervals (95%CI).

Ethical and biosafety aspects of research
The

RESULTS
Of the 194 male wild boars included in the study, three were discarded On the other hand, Table 1 shows the results of HEV in tissues of F I G U R E 1 Representative photomicrographs of tissue sections from wild boar naturally infected with hepatitis E virus (HEV), which was detected by real-time RT-PCR in serum. No immunohistochemical signals were observed in the liver (a), hepatic lymph node (b) and testis (c) of wild boar in which HEV RNA was not detected in these tissues. Meanwhile, in the naturally infected wild boar with HEV RNA detected by real-time RT-PCR in the different tissues, HEV antigen was also found in hepatic tissue (d), mainly in Kupffer cells (black arrow) and liver sinusoidal endothelial cells (black arrowhead). Likewise, in this animal, HEV was observed in macrophages (grey arrows) and cells with dendritic morphology (grey arrowheads) of the hepatic lymph node (e) and lightly in fibroblasts (white arrows) and some Sertoli cells (white arrowhead) of testis (f). Immunohistochemistry ( implications (Geng et al., 2016;Huang et al., 2018;Li et al., 2019).
A study carried out in China in infertile men showed a prevalence of HEV RNA genotype 4 in semen of 28.1%, with comparable viral titres in urine (Huang et al., 2018). In the same way, a study conducted in China including 26 semen samples collected from pigs found the Nevertheless, genotype 3c was detected in the ejaculate of chronically immunosuppressed HEV-infected men, even more than nine months after clearance of HEV viraemia (Horvatits et al., 2021). The difference in the results of these studies could suggest a relationship between the HEV genotype and the ability to produce testicular involvement and infertility. However, another study conducted in China did not con- and an increased necrospermia (Situ et al., 2020). All this gave rise to a transient infertility in the mice, which could partially be recovered after the complete elimination of HEV, whose permanence was longer in testis than in blood or faeces (42 vs. 28 days post-infection). Likewise, in animal models as Mongolian gerbils (Meriones unguiculatus), genotype 4 has also shown to induce molecular and structural changes in testis, damaging the blood-testis barrier (Soomro et al., 2017). In contrast, evidence of testicular damage in wild boars naturally infected with genotype 3 was not found in our study, even though HEV was detected in this organ by RT-PCR and IHC. Consequently, HEV genotype 3 infection does not seem to have a negative impact in fertility of male swine, as has been observed in female, with histopathological differences in the genitourinary tract between HEV genotypes. Similarly, it has been shown that HEV genotype 4 can replicate in the ovaries and promote apoptosis of oocytes  and genotype 1 has a high tropism for the placenta and decidua, inducing tissue apoptosis and necrosis (Gouilly et al., 2018), whereas genotype 3 showed a lack of tropism and induction of tissue damage in both structures.
This pilot study has some limitations that should be considered. The

CONFLICT OF INTEREST
The authors have declared no conflict of interest.

ETHICS STATEMENT
This study did not involve purposeful killing of animals. Professional personnel collected blood and liver samples mostly from hunted-

DATA AVAILABILITY STATEMENT
All data generated or analysed during the study are included in this published article. The datasets used and/or analysed during the present research project are available from the corresponding author on reasonable request. Sequences are available in GenBank under accession numbers OM525661, OM525662 and OM525663.